This invention relates to the fields of bio-organic chemistry, molecular biology, biochemistry, immunology and virology and human and veterinary medicine. In particular, it relates to methods and compositions for selective chemical modification of nucleic acids contained in compositions such as human blood, blood cellular components, blood plasma and plasma biopolymers purified from blood (albumin, clotting factors, immune globulin, fibrinogen etc.), cell culture constituents such as fetal bovine serum and porcine trypsin, non-blood products produced from normal or cancerous cells (e.g., recombinant DNA technology) such that each is rendered essentially free of infective viral contaminants and remains suitable for therapeutic or diagnostic use. In particular this invention also relates to methods of inactivating viral, bacterial, and cellular genomes in the preparation of killed vaccines and other medical products.
Transmission of viral diseases (e.g., hepatitis A and B, acquired immunodeficiency syndrome (HIV), cytomegalovirus infections) by blood or blood products is a significant problem in medicine. While donor selection criteria and screening of donor blood for viral markers helps reduce the transmission of viruses to recipients, screening methods are incomplete or less than 100% sensitive, as most are directed only to a few discrete viruses, and even in that case their sensitivity is insufficient. It is desirable to inactivate any virus contained in donor blood or blood products without altering the structure and function of its valuable constituents, e.g., red blood cells, platelets, leukocytes, and plasma biopolymers, such as proteins, polysaccharides etc. Similarly, other biological compositions, e.g., mammalian and hybridoma cell lines, products of cell lines, milk, colostrum and sperm, can contain infectious virus and it would be advantageous to inactivate said virus(es) while retaining the valuable constituents or products of these compositions. Finally, it is often unknown whether blood or blood products, or products of mammalian cells, contain infectious viruses. In this case it would be valuable to have compositions and methods to treat such cell- or biopolymer-containing composition to inactivate any infectious viruses present.
The manufacture of maximally safe and effective killed vaccines for human or veterinary use requires methods which completely and reliably render live microorganisms, e.g., viruses and bacteria, noninfectious ("inactivated") but which have minimal effects on their immunogenicity. Methods typically used for the inactivation of viruses, such as those useful in the preparation of viral vaccines, generally alter or destroy the function and structure of cells, cell components, proteins and other antigens.
Current inactivation methods, including the use of formalin, beta-propiolactone and ultraviolet radiation, have been developed empirically, with little basis in fundamental chemical or structural principles. Ethyleneimine monomers have been used to inactivate the foot-and-mouth disease virus (Russian patent no. SU 1915956). Ethyleneimine monomers have also been used to inactivate Mycoplasma and Acholeplasma (WO 92/18161) and avian infections (Romania patent no. RO 101400). Binary ethyleneimine has been used for inactivation of feline enteric coronavirus, FECV, (EP 94200383). Polyethyleneimine has been used as a plant virus control agent (JP 7882735). The foregoing methods and compounds modify microorganisms, such as viruses and bacteria, nonspecifically, and are difficult to standardize and apply reproducibly. In general, multiple components of the microorganism, including important surface antigenic determinants, for example viral capsid proteins, are affected by currently used inactivating agents, which modify not only nucleic acids but also other biopolymers such as proteins, carbohydrates and lipids, impairing their function. Altered antigens or inactivation of protective epitopes can read to reduced immunogenicity and hence low potency (e.g., inactivated polio vaccine), to altered antigenicity and hence immunopotentiation of disease instead of disease prevention (e.g., respiratory syncytial virus and inactived measles vaccines produced by formalin inactivation) or to appearance of new antigens common to another killed vaccine prepared with the same inactivant. Another example is in the preparation of a hepatitis B virus vaccine, where it is common practice to heat the preparation at temperatures in excess of 80.degree. C. and to treat with formaldehyde. These treatments not only inactivate viral infectivity, but also damage proteins and other antigens. Carrier substances added to the vaccine as stabilizers also may be unintentionally modified, producing allergic reactions, as occurs with human serum albumin in rabies vaccine inactivated with beta-propiolactone. In addition, ignorance of which chemical alterations render the microorganism noninfectious makes the process difficult to apply reproducibly. Periodic outbreaks of disease resulting from inadequate inactivation or reversion following inactivation are the result. Major outbreaks of paralytic poliomyelitis, foot and mouth disease and Venezuelan equine encephalitis have occurred due to this problem.
Hence none of the currently available agents used to produce inactivated viral vaccines are selective enough to completely inactivate infective viruses while preserving the antigenic properties of the virus particles, at least under conditions used so far for inactivation of the viral genome.
Yet another problem is that some of the viruses contaminating blood or other biological fluids are contained within the cell, either as a fully formed virus, viral genormle fragments or viral nucleic acid integrated into the host genome. For instance, the HIV virus is contained within leukocytes. It is a special concern to be able to inactivate both cell-free and cell-contained forms of virus, while retaining the structural integrity of cells.
The problems of inactivation of viruses in biological mixtures are distinct from the problems of inactivation of the viruses alone due to the co-presence of the desirable biopolymers such as proteins, carbohydrates, and glycoproteins in the plasma. While it is possible to inactivate the hepatitis B virus by using agents such as formaldehyde, or oxidizing agents, these methods are not suitable for the inactivation of the virus in blood due to the observation that most of these activating agents impair the biological activity of biopolymers in plasma or cellular components of blood. The use of ultraviolet light has been shown to inactivate viruses in a platelet concentrate. However, severe platelet damage resulted from higher doses. Beta-propiolactone reacts with nucleic acid and protein at similar rates; thus, while viruses can be inactivated, more than half of the factor VIII content of plasma is lost.
Problems may also exist in obtaining valuable biopolymers from non-blood sources since pathogenic viruses may also contaminate such compositions. These sources include, but are not limited to, mammalian colostrum and milk, ascitic fluid, serum, saliva, placental extracts, tissue culture cell lines and their extracts, including for example transformed cells, and products of fermentation.
It is an object of this invention to provide methods and compositions which allow selective modification of nucleic acid in the presence of other valuable biological macromolecules and cells. According to the methods and compositions of this invention, the nucleic acid of viruses, other microorganisms and cells, are selectively chemically modified, while preserving structure and function of non-nucleic acid components. It is also an object of the invention to provide selective inactivating agents to inactivate the virus, microorganism or cells while preserving their immunogenicity and achieving maximum reproducibility. It is another object of this invention to produce effective killed viral vaccines.